JP2003504025A - Delivery of trefoil peptide - Google Patents

Abstract

The present invention relates inter alia to use of a recombinant non-pathogenic and non-invasive bacterium transformed with DNA encoding a trefoil peptide and capable of delivering a trefoil peptide into the alimentary canal of a human or animal for the preparation of a medicament for treating lesions or inhibiting the formation of lesions of the alimentary canal caused by radiation therapy or chemotherapy for the treatment of cancer, a drug which damages the alimentary canal including alcohol, accidental exposure to radiation or to a caustic substance, infection, a digestive disorder including but not limited to non-ulcer dyspepsia, gastritis, peptic or duodenal ulcer, gastric cancer, MALT lymphoma, Menetier's syndrome or gastro-oesophageal reflux disease. The present invention also relates to a micro-organism, preferably a bacterial strain, preferably a non-pathogenic strain, preferably a non-invasive strain, preferably a food grade strain, preferably a gram-positive bacterial strain, delivering a trefoil peptide in vivo. Preferably said trefoil peptide is TFF1. The present invention further relates to a method for the delivery of trefoil peptide to the gastro-intestinal tract comprising the administration of such a bacterial strain. The present invention also relates to a pharmaceutical composition comprising a trefoil peptide delivering bacterium as well as methods of treatment of acute gastro-intestinal inflammatory diseases comprising administration of said transformed bacterial strains, particularly for treating acute colitis, including but not limited to acute flare-ups of Crohn's disease and ulcerative colitis in humans, as well as for treating gastro-intestinal disorders of a similar nature in other animal species.

Description

Detailed Description of the Invention

The present invention relates to the field of in vivo protein delivery systems. More particularly, the present invention relates to the secretion of trefoil peptides in vivo by a microorganism, preferably a strain, preferably a non-pathogenic strain, preferably a non-invasive strain, preferably a food grade strain, using the above system. The present invention relates to a method of delivering a trefoil peptide, and the use of the above trefoil peptide expression system for treating inflammatory disorders of the digestive tract.

Lactococcus lactis is a non-pathogenic Gram-positive lactic acid bacterium that can survive in the intestine (Klijn et al., 1995). L. l
It is uncertain whether actis is metabolically active in all of these environments.

Wells et al. (1993b) and Robinson et a.
l. (1997), Lactococcus l considering vaccination
Expression of tetanus toxin fragment C by actis has been described. further,
Either interleukin-2 or interleukin-6 was engineered to express with tetanus toxin fragment C (TTFC). It was demonstrated that when the lactis bacterial preparation was administered intranasally to mice, it produced 10 times more anti-TTFC than after similarly administering a strain expressing only TTFC (International Patent Application No. WO 97/14806). ). The above results allow the use of cytokine-secreting non-invasive experimental bacterial vaccine vectors to enhance the immune response to co-expressed antigens. Alternatively, the heterologous protein fragment is adhered to the inside of the cell wall, and in such a manner, the heterologous protein fragment is attached to the L. An approach has been described that appears on the lactis surface and is believed to lead to more enhanced vaccination properties (WO97094437, Steidler, Remout,
Wells).

Trefoil peptides are secreted by epithelial mucous cells and are stable in acidic environments. These peptides are thought to contribute to the protection of the mucosa (gel formation on the epithelium) and participate in the repair of mucous membranes damaged by stimulation of skin migration (Playford e).
t al. , 1996). Trefoil peptide production is increased locally at sites of injury, such as gastric ulcer and colitis (Wright et al, 19
90). Babyatsky et al. (1996) showed that administration of recombinant trefoil peptides reduced injury at these locations. In contrast to most other proteins important in mucosal protection, such as epidermal growth factor, in most studies trefoil peptides caused little or no proliferation (Playford et al, 1996). Three members of this trefoil peptide family have been identified in humans and were initially called pS2 (breast cancer estrogen inducible gene, 0.Lefebvre, 1993), SP (spasmolytic peptide) and ITF (intestinal trefoil factor). . The current nomenclature is p
S2 is renamed TFF1, SP is renamed TFF2, and ITF is renamed TFF3 (see, for example, Wong et al, 1999). Use this new designation throughout this text.

In humans, mice and rats, TFF1 and TFF2 are found mainly in the stomach and TFF3 is found mainly in the duodenum and colon. Wong et al. (1
999) provides a recent overview of trefoil peptides. The content of this document is incorporated into the present disclosure by reference.

[0006] TFF1 is thought to act through cell surface receptors (Tan et.
al, 1997).

WO 97/38712 and WO 92/14837 include mouth, esophagus, stomach,
The use of trefoil proteins or peptides for the treatment of disorders and damages of the digestive tract, including the large and small intestine, and for the protection and treatment of tissues outside the digestive tract has been described. These proteins can be used either to treat lesions at these sites or to inhibit lesion formation. These lesions may be due to radiation or chemotherapy to treat cancer, inadvertent exposure to other drugs, including alcohol that damage the digestive tract, radiation or caustic substances, infections, chemicals, bacteria or unknown causes. , Non-ulcer dyspepsia, gastritis, peptic ulcer, duodenal ulcer, gastric cancer, MALT lymphoma, Menetier's syndrome, gastroesophageal reflux disease,
It may be due to dyspepsia, including but not limited to Crohn's disease, ulcerative colitis and acute colitis.

[0008] Trefoil peptides are particularly useful in the treatment of acute colitis.

ITF is also used in combination with EGF (epithelial growth factor) to treat gastrointestinal ulcers. In vitro and in vivo experiments, it was demonstrated that the combined treatment of EGF and ITF significantly increased the wound healing activity of EGF without enhancing the proliferative effect of EGF (Chinery and Playford.
, 1995).

Inflammatory bowel disease is a group name for a range of gastrointestinal inflammations. Belonging to this group are enteritis, colitis, inflammation of the mucosa of the duodenum or colon, respectively. Crohn's disease, enteritis reg
ionicis and ulcerative colitis
, Colitis ulcerosa) is a closely related, chronic and naturally recurring gastrointestinal tract disorder. Diseases are immunologically mediated and have environmental and genetic causes. Sartor (1995) describes different aspects of inflammatory bowel disease. Crohn's disease can be described, for example, in Herfath and
Sartor (1994), Cominelli et al. (1994), and Ma.
It has been extensively studied by cl Dermott (1989).

An object of the present invention is to provide a method of delivering trefoil peptides for treating gastrointestinal disorders.

Another object of the present invention is to provide a medical composition for treating gastrointestinal disorders.

More particularly, the present invention relates to microorganisms that deliver trefoil peptides in vivo. Preferably, the microorganism is a strain, preferably a non-pathogenic strain, preferably a non-invasive strain, preferably a food strain, more preferably a Gram-positive strain, most preferably lactic acid. Fermentative strains, preferably Lactococcus species or Lactobacillus species that express trefoil peptides in vivo. Therefore, the present invention provides

[0014]

[Table 1]

It can be applied to any of the Lactococcus species or Lactobacillus species or subspecies selected from the list comprising

Due to the ability of Lactococcus lactis to deliver one heterologous antigen, or to produce molecules such as IL-2 and IL-6 in vitro and in vivo, Lactococcus lactis is expressed in vivo.
It was not clear that o was a suitable vehicle to deliver other types of peptides or polypeptides. Furthermore, it is unclear whether the trefoil peptides expressed by the strains have a beneficial effect on inflammatory diseases of the digestive tract, such as inflammatory bowel disease or acute colitis.

Therefore, it can be clarified in the Examples section of the present specification that the strain, when present in the digestive tract, can express the trefoil peptide in vivo and exert a curative effect in the state of acute colitis. I'm surprised. As an example, a PCR fragment containing the coding region mouse TFF1 was cloned. A recombinant vector containing these PCR clones under the control of a promoter and the usp45 Lactococcus lactis secretion signal sequence was constructed. Mouse T
Transformed Lactococcus la expressing FF1 trefoil peptide
The ctis strain was constructed. Furthermore, recombinant mTF produced by these bacteria
It was demonstrated in an in vivo mouse model system that F1 surprisingly exerts a healing effect on the distal part of the inflamed colon.

According to a preferred embodiment, the invention relates in particular to strains which deliver the trefoil peptide in vivo.

According to another preferred embodiment, the invention relates to a bacterium that delivers TFF1 in vivo.

It should be understood that the present invention also relates to some or variant forms of the trefoil peptides. The above portion can be generated by methods well known to those skilled in the art,
Refers to the biologically active portion. These moieties are generally at least 10 consecutive amino acids, generally at least 20 consecutive amino acids, more usually at least 30 consecutive amino acids, usually at least 40 consecutive amino acids, preferably at least 50 consecutive amino acids. Contains individual amino acids. Said variant refers to a variant having the same biological activity as the trefoil peptide described above.

It will also be clear that the strains according to the invention described above may also express additional recombinant proteins useful in the treatment of the expected disorders.

According to yet another embodiment, the present invention relates to a medical composition comprising a microorganism expressing the trefoil peptide described above.

Preferably, the medical composition according to the invention is conveniently suitable for use on mucosal surfaces.

According to the invention, the medical composition for use according to the invention comprises, in addition to microorganisms, pharmaceutically acceptable excipients, carriers, buffer solutions, stabilizers or other substances known to the person skilled in the art. It may also contain substances. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration. The person skilled in the art is perfectly able to prepare suitable solutions.

According to another embodiment, the invention relates to a method of delivering a trefoil peptide to the gastrointestinal tract, comprising the administration of a microorganism as described above.

According to another aspect, the invention also relates to the use of the above-mentioned microorganism for the manufacture of a medicament for delivering a trefoil peptide to the digestive tract.

According to another embodiment, the present invention relates to a method for the treatment of gastric and / or intestinal diseases and / or disorders, which comprises the administration of microorganisms of the aforementioned microorganisms.

The present invention also relates to a method of treating gastric and / or intestinal diseases and / or disorders comprising the administration of a microorganism that delivers a TFF1 trefoil peptide in vivo.

The trefoil protein expressed by the strain according to the invention can be used both for the treatment of lesions at these sites or for the inhibition of lesion formation due to gastrointestinal diseases and disorders.

The expression “gastric and / or intestinal diseases and / or disorders” relates to any type of stomach, intestine and gastrointestinal diseases and / or disorders. In a preferred embodiment of the invention, this phrase relates to acute gastrointestinal inflammatory diseases and disorders. These diseases are preferably acute gastrointestinal disorders of chemical, bacterial or unknown origin. Belonging to this group are enteritis, colitis, including but not limited to acute recurrence of Crohn's disease and ulcerative colitis inflammation of the mucosa of the duodenum or colon, respectively. This includes traveler's illness. In another preferred embodiment, the expression "stomach and / or intestinal disease and / or disorder" according to the invention relates to chronic and spontaneous recurring gastrointestinal diseases such as Crohn's disease and ulcerative colitis.

The expression “stomach and / or intestinal disease and / or disorder” also relates to diseases involving lesions on the mucosal surface. Thus, disease states treated by the methods and compositions of the present invention may include gastrointestinal disorders and damage including the mouth, esophagus, stomach, large intestine and small intestine, and protection of tissues outside the gastrointestinal tract. And also for treatment. These lesions may be due to radiation or chemotherapy to treat cancer, inadvertent exposure to other drugs, including alcohol that damage the digestive tract, radiation or caustic substances, infections, chemicals, bacteria or unknown causes. , Non-ulcer dyspepsia, gastritis,
Peptic ulcers, duodenal ulcers, gastric cancer, MALT lymphomas, Menetier's syndrome, gastroesophageal reflux disease, and Crohn's disease can result from non-limiting digestive disorders.

The invention therefore relates to the use of the abovementioned microorganisms for preparing a medicament for treating gastric and / or intestinal diseases and / or disorders.

The present invention is intended to treat acute gastrointestinal inflammatory diseases, acute colitis, acute recurrence of Crohn's disease and ulcerative colitis, as well as chronic and spontaneous recurrences including Crohn's disease and ulcerative colitis. It also relates to the use of the above-mentioned microorganisms for the preparation of a medicament for treating diseases of the digestive tract.

According to another embodiment, the present invention relates to the use of the above-mentioned microorganism for preparing a medicament for suppressing the formation of lesions due to gastric and / or intestinal diseases and disorders.

The administration of the microorganism may be oral, or other methods known in the art that allow the microorganism to enter the desired location to be treated, eg anal administration,
It may be vaginal administration. The microorganisms are in culture, ie (at least in vivo).
It can be used in a medium containing substances that maintain the metabolic activity of the microorganism (in tro). Such substances maintain viability even if they do not maintain microbial growth. Such substances may include energy sources such as glucose and amino acids.

[0036]   The individual to which the microorganism is administered may be human or animal.

In therapeutic situations, ie, where the biological effect of delivering the polypeptide to an individual is beneficial to that individual, administration is preferably a “therapeutically effective amount”,
This is sufficient to show benefit to the patient. Such benefit may be an improvement in at least one symptom. The actual dose, rate of administration and time course of administration will depend on the purpose of administration, eg the biological effect sought in view of the nature and severity of microbial administration and is a matter of routine optimization. Prescription of treatment, eg decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

The composition comprising the microorganism according to the invention may be administered alone according to the invention or in combination with other treatment methods, either simultaneously or sequentially.

According to another embodiment, the present invention uses a recombinant vector carrying a trefoil polypeptide coding sequence under the control of a suitable promoter and a suitable bacterial secretion signal sequence, as described above. A method of making a microorganism that delivers a trefoil peptide in vivo comprising transforming the microorganism.

The bacterial secretion signal sequence may be any sequence known in the art to perform the above actions. L. In the case of Lactis, the secretion signal is usp45 L. It is preferably a lactis secretion signal sequence. The promoter sequence may be any promoter capable of expressing the coding sequence in the microorganism. Examples shown in the Examples section include known inducible E. E. coli phage T7 promoter and L. Included is the known component P1 promoter of lactis.

The present invention also relates to a recombinant vector comprising at least part of a trefoil peptide coding sequence under the control of a suitable promoter and a suitable secretory signal sequence. Using the above recombinant vector, at least a part of the trefoil peptide sequence capable of exerting a healing effect on the damaged site on the membrane surface is in vivo.
can be delivered vo.

The present invention further relates to the above-described recombinant vector having a nucleotide sequence represented by any of SEQ ID NOs: 1, 2 or 4.

The following examples serve only to illustrate the invention by way of example and should not be considered to limit the invention in any way.

[0044]   All materials mentioned in this document are incorporated herein by reference.

[0045]

【Example】

Example 1: Cloning and expression medium of mouse TTF1 (mTTF1) GM17 was supplemented with 0.5 w / v% glucose in M17 (Difco, Detro.
It). The M9 medium contains the following per liter.
Na ₂ P0 ₄ 6 g, KH ₂ PO ₄ 3 g, NH ₄ Cl 1 g, NaCl 0.5 g
, MgSO ₄ 2 mmol, CaCl ₂ 0.1 mmol and Casitone (Dif
co) 5 g. M9B contains 2.1 g of NaHCO ₃ and Na ₂ C per liter.
2.65 g of O ₃ is added to M9. GM9B is 0.5 w / v% glucose added to M9B. LM9B contains 0.5 w / v% lactose in M
It is added to 9B.

Where applicable, the antibiotics erythromycin (Er) or chloramphenicol (Cm) were added to each medium at a final concentration of 5 μg / ml, respectively. The nomenclature used to indicate the presence of antibiotics is eg GM17Er, LM9BCm etc. The solid medium contained 1.2% agar.

Recombinant DNA Technology DNA modifying enzymes and restriction endonucleases were used under standard conditions in the buffer recommended by the manufacturer. General molecular cloning techniques and electrophoresis of DNA and proteins were performed according to standard procedures. By electroporation of cells grown in the presence of glycine, L. Lactis was transformed. (Wells et al., 1993a). The plasmid DNA is Q
Purified as usual using the iagen Plasmid Kit.

PCR amplification of mTFF1 Using the oligonucleotide primers mTFF1S and mTFF1A,
PCR reaction was performed on a plasmid containing mTFF1 cDNA (Lefe
bvre, 1993). The mTFF1S primer corresponds to the first 18 nucleotides after the signal sequence to the first 18 nucleotides of the sense strand of mTFF1.
The mTFF1A primer contains the last 2 of the sense strand of mTFF1 including the stop codon.
It is complementary to 6 nucleotides and introduces a special Spel restriction site.

[0049]

[Table 2] Here, ACTAGT in mTFF1A represents a Spel site.

Vent ^™ DNA Polymerase (N
Using ewEnglish Biolabs (Beverly, USA),
PCR amplification was performed. The PCR mixture consisted of 2 units of Vent DNA polymerase, 10 μl of Vent buffer solution (thermopol), dXTP.
4 μl (maximum 0.5 mM), 5 μl of each primer (0.5 μM), template DNA 1
It consisted of μl (50 ng) and 74 μl of H ₂ O. 0, 1, 2 respectively
Six reactions were prepared with different final concentrations of MgSO ₄ , adjusted to 3, 4 and 5 mM. The PCR amplification cycle was as follows. T ₀ : 94 ° C. for 300 seconds,
T ₁ : 94 ° C. for 45 seconds, T ₂ : 60 ° C. for 30, T ₃ : 72 ° C. for 20 seconds, T ₄ : 20 ° C.
10 seconds. These cycles from T ₁ to T ₃ were performed 30 times. PCR amplification with these primers gives the gene for mature mTFF1 lacking the signal sequence and containing an additional Spel restriction site. After confirmation by gel electrophoresis, the amplified fragments appear as bands in the expected length range. mT
The 5'end of the FF1 sequence contains two possible target sequences complementary to the forward primer. As a result, the above-mentioned primers can be used to amplify two fragments of 202 and 208 base pairs respectively from mTFF1 cDNA. It is considered that these fragments cannot be analyzed by agarose gel electrophoresis.

Plasmid Construction Two different types of vectors were used as acceptors for the mTFF1 trefoil peptide encoding the PCR fragment. Two parental vectors (pTR
PT1NX derived from EX1 (Wells and Schofield, 1996), and pLET2NX (Steidler et al, 1995)
) Derived pLET2NX) contains the following common elements: promoter (T7 or P1), the sequence encoding the ultimate amino acid residue (
Steidler et al, 1995), modified to include a Nael restriction site that overlaps. lactis usp45 secretion signal sequence (van
(European patent application issued under Asseldonk et al, 1990 and 0455280) and downstream Spel restriction sites. The pT1NX-derived plasmid identifies resistance to erythromycin and the pLET2NX-derived plasmid identifies resistance to chloramphenicol. DNA solution 5
0 μl, Spel buffer solution 10 μl, Spel 50 units, T4 polynucleotide kinase (Gibco BRIL, Bethesda, USA) 10 units, pH
PCR using 0.5 mM ATP adjusted to 7.5 and 36 μl H ₂ O
The fragment was treated at 37 ° C for 1 hour. Purified DNA 10-20 μl, Nae
l buffer solution 10 μl, Nael 10 units, Spel 50 units, calf intestinal alkaline phosphatase (Boehringer, Mannheim, Germany)
y) 37 units of vector pT1NX with 1 unit and 73-63 μl of H ₂ O.
Digested at ° C for 1 hour. After 30 minutes of incubation, 50 units of Spel and 10 units of Nael were added back to the mixture. The restriction enzyme was inactivated and the mixture was extracted with phenol / chloroform extraction. After restriction digestion, "mouse TFF1
(MTFF1) PCR Amplification ”, and the band derived from mTFF1 (
The 195 bp and 201 bp fragment) and the vector portion were excised from the agarose gel. "Ready To Go" T4 DNA ligase (
Recombinant plasmid containing mTFF1 cistron as an in-frame fusion to the usp 45 secretory signal sequence under the control of the promoter after ligating each PCR fragment with the vector for 45 minutes at 16 ° C. (Pharmacia Biotech, UK). was gotten.

Component L. Plasmid pT1mTFF containing the lactisP1 promoter
1 (FIG. 1a) shows the purified Nael-Spel vector part of pT1NX and Spe.
It resulted from ligation with a 1-cut and 5'phosphorylated PCR fragment.

Inducible E. Plasmid pL2m containing the E. coli phage T7 promoter
TFF1v1 (FIG. 1a) resulted from the ligation of the purified Nael-Spel vector portion of pLET2N with the Spel cleaved 5 ′ phosphorylated PCR fragment. The T7 promoter can only be activated by, for example, the cognate T7 RNA polymerase encoded by the plasmid pILPOL.
This plasmid Lactis strain MG1820 [pILPOL] (Wells et al, 1993c).

For structural analysis, the plasmid pT1mTFF1 was transformed into L. lactis strain MG13
Transformed into 63. Cells were grown on GM17Er plates. GM17Er
Colonies were grown in 2.5 ml and plasmids were isolated. PT1NX vector (2 μl of DNA (pT1NX), 20 units of EcoRI, 50 units of Spel, 2 μl of Spel-buffer solution and 15 μl of H ₂ O) and isolated recombinant plasmid (5 μl of DNA, 20 units of EcoRI) using analytical digestion. Spel
50 units, 2 μl of Spel-buffer solution, 0.2 μl of 10 μg / ml Rnase A stock solution
The restriction patterns of 5 μl and H ₂ O 12 μl) were compared. This plasmid is Eco
It cut | disconnected by RI and Spel at 37 degreeC for 1 hour. 9 for the reference plasmid
Two linear fragments of 07bp and 4999bp are predicted. pT1m
For TFF1, two bands of 499 bp and 4999 bp are predicted.
The size of the experimentally obtained fragment visualized by agarose gel electrophoresis and EtBr staining was consistent with the expected length. 1 from each recombinant plasmid
One positive culture was streaked on a GM17Er plate to obtain isolated colonies. Subsequently, one colony was inoculated into 100 ml of GM17Er medium and grown to saturation. The cells were harvested and the plasmid was purified. Their physical structure was verified by restriction enzyme analysis and agarose gel electrophoresis. Furthermore, sequence analysis revealed that mTFF1
It was revealed that the cistron was completely ligated in frame with the usp45 secretory leader sequence. pT1mTFF1 contains a 208 bp insert representing the entire coding sequence of mature mTFF1 (“PCR of mouse TFF1 (mTFF1).
Amplification ”mentioned above).

For structural analysis, plasmid pL2mTFF1v1 was used as strain MG1820 [pIL
POL]. Cells were grown on GM17Cm plates. GM17
Colonies were grown in 2.5 ml Cm and plasmids were isolated. Using the analytical digest, pLET2NX vector (DNA (pLET2NX) 2 μl, Eco
RI 20 units, Spel 50 units, Spel-buffer solution 2 μl and H ₂ O
15 μl) and the isolated recombinant plasmid (DNA 5 μl, EcoRI 20 units, Spel 50 units, Spel-buffer solution 2 μl, 10 μg / ml Rnase).
The restriction patterns of A stock solution 0.25 μl and H ₂ O 12 μl) were compared. This recombinant plasmid was digested with EcoRI and Spel at 37 ° C for 1 hour. For the reference plasmid, two linear fragments of 907 bp and 4650 bp are predicted. For pL2mTFF1, two bands of 499 bp and 4650 bp are predicted. Visualized by agarose gel electrophoresis and EtBr staining,
The size of the experimentally obtained fragment was consistent with the expected length. From the recombinant plasmid, one positive culture was streaked on GM17Cm plates to obtain isolated colonies. Subsequently, one colony was inoculated into 100 ml of GM17Cm medium and grown to saturation. The cells were harvested and the plasmid was purified. Its physical structure
It was verified by restriction enzyme analysis and agarose gel electrophoresis. In addition, sequence analysis revealed that the mTFF1 cistron was ligated in frame with the usp45 secretory leader sequence. pT1mTFF1 contains a 208 bp insert representing the entire coding sequence for mature mTFF1 (described above under “PCR amplification of mouse TFF1 (mTFF1)”). This analysis also shows that pL2mTFF1v1 is 2
It was shown to contain a 02 bp insert (thus lacking the first two amino terminal amino acid residues of mature mTFF1; "mouse TFF1 (mTFF1) PC".
R amplification ”above). The sequence of the recombinant plasmid is shown in Figures 1b and 1c. All these sequences were compiled from the published component sequences. Furthermore, PCR
Relevant sections of sequences such as fragments and ligation junctions were verified experimentally.

Transformation L. Protein expression in lactis Using the plasmid constructed as described above, L. The Lactis strain was transformed. For transformation of the pT1mTFF1 plasmid, L. lactis strain MG
1363 (Gasson, 1983) was used. For transformation of the pL2mTFF1v1 plasmid, L. lactis strain MG1820 (pILPOL) (M
aeda and Gasson, 1986) was used.

These transformed L. The expression of the protein by the Lactis strain was determined by SDS-
It was detected by PAGE.

To prepare the culture supernatant fraction, GM1 in the case of the pT1mTFF1 plasmid
In 5 ml of 7Er medium or GM1 for pL2mTFF1v1 plasmid
Cells were grown for 20 hours at 28 ° C in 5 ml of 7Cm medium. Culture the GM17
Dilute 1/100 with 5ml of either Er medium or GM17Cm medium, 28 ℃
Were grown for 3 hours. The cells are harvested by centrifugation at 2800 rpm for 20 minutes and the appropriate medium, ie GM9BEr for MG1363 cells,
Alternatively, in the case of MG1820 [pILPOL] cells, the cells were resuspended in 5 ml of LM9BCm. After growing for an additional 5 hours, the cells were pelleted. Proteins present in the media fraction were removed by phenol extraction and ethanol precipitation.

[PiLPOL; pL2mTFF1v1] transformed with L. lact
The L. is MG1820 strain and L. p.1 transformed with [pTREX1; pT1mTFF1]. L. lactis in comparison to Lactis MG1363. lactis MG
The proteins expressed in the culture supernatant fraction of the 1820 control strain are shown in FIG. This figure shows MG1820 [pL2mTFF1v1] and MG1 when compared to controls.
Shown is a special protein band (indicated by edging) of appropriate size in 363 [pT1mTFF1]. As can be seen from this figure, the recombinant gene expression is quite low. Others have used dramatically higher levels of purified trefoil peptides in treatments to repair gastric and intestinal damage (eg, Tran et al. (1999) at daily levels of 2.5 mg / kg body weight). Confirmed because 5 days of rectal administration of human recombinant TTF2 was used to reduce the inflammatory index of colitis (intestinal administration of dinitrobenzenesulfonic acid dissolved in alcohol) in experimentally-incorporated rats. The in vivo results were surprising.

Example 2: In vivo test of MG1363 [pT1mTFF1] Cell preparation for intragastric administration L. lactis strains MG1363 [pTREX1], MG1363 [pT1
The transformant of [mTFF1] was streaked on a GM17Er plate and grown overnight at 28 ° C. In each case one colony was grown overnight at 28 ° C. in 15 ml of GM17Er medium. To store the cells at -20 ° C, add 100 to this culture.
15 ml of% glycerol was added. It was possible to inoculate the required amount of cells daily to treat mice. To this end, the culture was diluted 1/200 in 10 ml of GM17Er medium. After growing at 30 ° C. for a minimum of 20 hours,
Cells were harvested by centrifugation at 2800 rpm for 15 minutes. The cells were then resuspended in 1 ml M9B without antibiotics.

In vivo studies in mice with acute colitis. The effect of trefoil peptides expressed from Lactis bacteria was tested in mice with acute colitis. Female Balb / c mouse 21 during 7 days
5% DSS (dextran sulfate sodium) dissolved in drinking water was administered to the animals. In this way, acute colitis was induced (Koujoharoff et al,
1997). For treatment, from 1 to 7 days of DSS treatment, 100 bacterial suspensions
These mice were orally inoculated daily with a gastric catheter using μl (minimum 1.10 ⁸ cells). As directed, MG 1363 [pTREX1] was administered to 6 mice.
6 cells were inoculated with MG1363 [pT1mTFF1] cells,
Three mice were not inoculated (DSS control). Mice were sacrificed 8 days after induction of colitis and examined immunologically and histologically.

Immunological tests of sera revealed that treated mice did not show an immune response to the expressed protein. Serum was collected from mice exsanguinated on the 8th. This serum is It was analyzed by Western blotting to see if it contained antibodies to the proteins present in the medium fraction of Lactis cells. The medium fraction used was L. lactis strains MG1363 [pTREX1] and MG
It was derived from 1363 [pT1mTFF1]. Equivalents of 1 ml of concentrated (phenol extracted and ethanol precipitated) medium fraction were analyzed by SDS-polyacrylamide (20%) gel electrophoresis. After blotting on nitrocellulose filters, the filters were incubated with 4 groups of mouse serum solutions for 1 hour. Nitrocellulose blocking buffer solution (Blotto: 10 x PBS)
The serum was diluted 500-fold with 100 ml, 150 ml of 1 M NaCl, 2 ml of Triton X-100, 25 g of nonfat dry milk, 20 ml of water up to a total dose of 1 liter). As a secondary antibody, sheep anti-mouse IgG conjugated with horseradish peroxidase (HRP) was used. No signal was detected using serum diluted 500-fold.

Histological analysis was performed on the colons of treated mice. The colon was cut longitudinally and bisected into a distal portion (closest to the anus), an intermediate portion and a proximal portion. These colon sections were histologically analyzed after fixation with 3.7% formaldehyde (in PBS) overnight to ensure tissue sample standing in paraffin blocks and paraffin embedding. From each tissue sample, three parallel long axis sections with a thickness of 3 μm were made uniformly spaced throughout the sample. These cross sections were colored with hematoxylin / eosin. Prior to scoring sections, histological analysis was performed in a blind fashion, which meant covering the label on the slide. Slides bearing sections from several groups of mice were randomized and then examined. Each slide was then assigned a histological score (0 to 5) according to the symptom description in Table 1.
Range).

An average score of 3 sections was calculated for each mouse and each colon section. Inflammation, consisting of epithelial damage and invasion, was most prominent in the distal and middle parts of the colon. Almost no inflammation was observed in the proximal part. Mean histological scores were calculated for both the distal and mid colon sections per group of animals. The final histological total score is the sum of two separate scores (total score =
Epithelial damage score + invasion score), a measure of the degree of inflammation. The total histological score of the distal colon section for each group of mice is shown in FIG.

From the histological scores for the distal portion of the colon shown in FIG. One could conclude that when mice were inoculated with Lactis cells, inflammation was significantly reduced. [PT1mTFF1] Transformation L. l
Mice administered actis cells show a significant reduction in inflammation of greater than 65%.

As can be seen in FIG. 3, inflammatory infiltrates and epithelial damage in the distal portion of the colon are associated with recombinant L. cinerea secreting mTFF1 polypeptides. It was significantly reduced by inoculation with the Lactis strain.

The above results were confirmed in another experiment carried out in the same way, which included a larger group (group size = 10) and more control groups. FIG. 4 shows healthy control mice (control) and DSS administered as described and left untreated (
DSS) or MG1363, MG1363 [pT1TREX1 as instructed
] Or MG1363 [pT1mTFF1] treated mice (as described above) with histological scores (obtained as described above). From the experiment, MG1363 [pT1m
A clear and marked reduction of intestinal inflammation is seen in the group of mice treated with TFF1].

The latter experiment was performed by determining the levels of interleukin-10 (IL-10) and interferon-γ (IFN-γ), both of which are proinflammatory cytokines well known to those skilled in the art. Also evaluated. Mice (n = 10) were inoculated with the strain as indicated. Control = healthy mouse, DSS = 5% DS dissolved in drinking water
Mice that received S and were not treated. The colon was prepared and a punch (diameter = 4 mm) was used to isolate a section with the same surface. RP on the tissue sample of each group
500 μl of MI + 10% fetal bovine serum was added and incubated at 37 ° C. overnight. The supernatant was collected and titrated for cytokine content by ELISA. IL- in each tissue
The amounts of 1β and IFN-γ are shown in FIG. From the results, MG1363 [pT1mT
A clear reduction of these pro-inflammatory cytokines in the group of mice treated with FF1] can be seen.

Example 3: Comparative plasmid construction for treatment with MG1363 [pTITFF1] and purified TFF1 In case of expression of mTFF1 form Pichia pastoris, plasmid pP.
ICmTFF1 was constructed. For this, a description (PCR amplification of mouse TFF1)
The mTFF1 gene was PCR amplified as usual. This fragment is called pPIC9
A derivative of (Invitrogen) was ligated with the exposed Nael restriction site. The ligation mixture was transformed into E. E. coli MC1061 transformed and correctly assembled clones were identified by restriction analysis and DNA sequencing (sequence in Figure 1d). In the plasmid pPICmTFF1 thus obtained, the mTFF1 sequence is fused in frame with the Sacharomuces cerevisiae α-mating factor preprosecretion signal.

Expression and Purification of mTFF1 Plasmid pPICmFF1 was transformed into P by the method described in Logghe (1995).
A positive clone transformed with ichia pastoris GST115 and having the mTFF1 unit integrated at the his4 locus was selected by PCR identification. These positive clones were induced with methanol and screened for expression by protein analysis of the culture supernatant and compared to a negative control (negative), 6.5.
One clone showing a particularly high expression of a particular band in GDa (GST115 :: pPICmTFF1) was kept for further study (FIG. 6, marked by edging). A particular protein band was identified by protein sequencing as mTFF1. The expression procedure was optimized and scaled up to a 16 liter culture and mTFF1 was purified from the culture supernatant. To this end, methanol-induced GST
1 15 :: pPICmTFF1 supernatant was tangentially filtered (Millipore pro).
Flux M12, cut off at 3000 Da) and concentrated to 0.02 pH 7.4.
It was dialyzed against M phosphate buffer solution. Ion exchange column (Biorad's Q-column)
Was used to purify mTFF1 from this concentrate. The protein was eluted from the column by an isocratic salt gradient. The mTFF1 thus obtained was> 99% pure and was further concentrated. The final preparation contains less than 160 ng / ml LPS. This amount of LPS is within acceptable limits and the pS2 protein can be used in further experiments. Purify mTFF1 with a size exclusion column (
After analysis with Superdex 75; Pharmacia), it was concluded that 7.5% of mTFF1 was in the monomeric form (Fig. 7A). This was confirmed by reducing SDS-PAGE and non-reducing SDS-PAGE of purified mTFF1 (FIG. 7B).

Evaluation of the biological activity of purified TFF1 A well-known feature of the TFF1 protein is the markedly reduced surface expression of E-cadherin after administration of this protein to Caco-2 cell monolayers (Liu.
et al, 1997). We have shown a 10% reduction in E-cadherin surface expression after administration of the mTFF1 preparation described above to Caco-2 monolayers.

Treatment of murine acute colitis with purified mTFF1: To induce acute colitis, 6% dextran sulfate sodium (DSS, MW40000) dissolved in drinking water was administered to mice for 7 days (Kojouhar).
off et al. , 1997). The mice used in the experiments were age-matched and simultaneously received DSS administration. DSS from -7 to 0 days for treatment
50 μg of mTTF1 dissolved in 200 μl of PBS before administration (pre-administration group), from 0 to 7 days, during DSS administration (administering group), and from 7 days to 14 days after administration (post-administration group) Was administered to mice. To study different routes of delivering mTFF1, mice were administered by ip injection, intragastric inoculation and rectal administration in each setting. After the DSS-free drinking water was given for 1 day, the mice were sacrificed on the 8th day (the pre-administration group and the administration group), and the DSS-free drinking water was given for the 7th day, and then on the 14th day They were sacrificed (post-administration group). DS dissolved in drinking water
The untreated control group that received S was sacrificed on days 8 and 14. All groups consisted of 9 mice. The results shown in FIG. 8 clearly show that no statistically significant improvement was observed in any treatment plan. The described invention was surprising since an improvement was identified (Figures 3 and 4). This is the L. T by lactis
Delivery of FF1 is meant to contribute essentially to the observed therapeutic effect.

[0073]

[Table 3]

[0074]

[Table 4]

[Brief description of drawings]

FIG. 1: Schematic representation of the plasmids used. FIG. 1a: Schematic map of plasmids pL2mTFF1v1 and pT1mTFF1. T7 is the major late promoter from coliphage T7. (Stud
ier and Moffatt, 1986). P1 is Waterfield
(1995), where usp45S is a DNA fragment encoding a secretory signal peptide derived from the lactococcal Usp45 protein (vanAsseldonck et al, 1990), and mT
FF1 is a DNA fragment encoding the mature part of murine TFF1,
mtff1v1 is a DNA fragment encoding a truncated (lacking two amino terminal amino acid residues) mature murine TFF1, Cm is a chloramphenicol selectable marker, and Em is an erythromycin selectable marker. pPI
About CmTFF1: PPMF is a pre-pro Saccharomyces ce
revisiae α-mating factor, AOX1 prom is an alcohol oxidase promoter, AOX1 term is an alcohol oxidase terminator, HIS4 is a histidol dehydrogenase gene, Ori is an Escherichia coli origin of replication, and AOXfr is an alcohol oxidase. Is the 3'fragment of the gene, A
mpR is the ampicillin resistance gene and all components are derived from the pPIC9 plasmid (Invitrogen).

Figure 1b   Figure 1b: DNA sequence of plasmid pL2mTFF1v1 (SEQ ID NO: 1).

[Fig. 1c]   Figure 1c: DNA sequence of plasmid pT1mTFF1 (SEQ ID NO: 2).

[Fig. 1d]   Figure 1d: DNA sequence of plasmid pPICmTFF (SEQ ID NO: 3).

FIG. 2: SDS-PAGE. The different protein fractions are from L. lactis MG
1820 [pILPOL] (control), MG1820 [pILPOL; pL2mT
FF1v1], MG1363 [pTREX1] or MG1363 [pT1mT
FF1] Derived from the cell culture medium. The two lanes on the left contain a marker protein whose molecular weight is given in kDa. This protein was visualized using Coomassie blue staining.

FIG. 3: Display of histological scores of the distal part of the colon. Upper left graph: epithelial damage (distal partial colon). Upper right graph: inflammatory infiltrates (distal partial colon). Lower graph: upper (
Distal partial colon) total histological score.

FIG. 4: Healthy mice (control) or untreated (DSS) or M
G1363, MG1363 [pTREX1] cells or MG1363 [pT1m
Display of histological scores of the distal portion of the colon of mice with acute DSS colitis after treatment with TFF1] cells.

FIG. 5: Proinflammatory cytokine titration in acute inflamed colon tissue. Healthy mice (control) or untreated (DSS) or MG1363, MG13
Interleukin-1β in the distal colon of acute DSS colitis mice after treatment with 63 [pTREX1] cells or MG1363 [pT1mTFF1] cells
(Left) and interferon-γ in the middle and distal colon (right).

FIG. 6: Selected Pichia pastoris (GST115 :: pPI
CmTFF1) media-derived protein fraction and negative control SDS-PAGE
. In addition, the mTFF1 producer clone used for the production of mTFF1 is shown in tandem. This protein was visualized using Coomassie blue staining.

FIG. 7A: Gel filtration pattern of purified mTFF1 (Superdex75; Pharmacia). The mTFF1 protein elutes in two peaks, the majority of which are present in fractions 14, 15, 16 (dimers) and 20 (monomers). The identity of the protein in these fractions proved to be mTFF1 by SDS-PAGE (inset). This protein was visualized using Coomassie blue staining. FIG. 7B: SDS-PAGE of purified mTFF1 under reducing and non-reducing conditions. The left lane is a size marker of the indicated size (Coomassie Brilliant Blue stain).

FIG. 8: Acute DSS-induced colitis pre-integration (pre), during (du) or post-integration (po), intraperitoneal injection (ip), oral (oral) and rectal inoculation. Display of histological scores of the distal part of the mouse colon treated by (rectal) inoculation. DSSdu represents the score of mice that received PBS to induce acute DSS colitis.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12N 15/00 ZNAA // (C12N 1/21 A61K 37/02 C12R 1:01) (81 ) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG) , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, B R, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Stadler , Lotar Belgium, Bee-9160 Lokeren, Bockslarstrat 41 (72) Inventor Remout, Eric Rehne Belgium, Bee-9921 Ronendehem, Belfstraat 7 F term (reference) 4B024 AA01 BA80 CA04 CA20 CA05 DA12 EA04 FA02 FA18 GA11 HA03 4B065 AA01X AA77X AA91Y AB01 AC14 BA02 BA24 BB01 BC03 BD14 CA24 CA43 CA44 4C084 AA02 AA13 BA44 CA53 CA56 MA56 NA14 ZA681 4C087 AA01 BC55 BC56 CA12 MA52 MA56 NA14 ZA66

Claims (18)

[Claims] 1. A recombinant microorganism that delivers a trefoil peptide in vivo. 2. The microorganism according to claim 1, wherein the microorganism is a strain. 3. The microorganism according to claim 2, wherein the microorganism is a food-grade strain, preferably a Gram-positive strain. 4. The microorganism according to claim 3, wherein the strain is a Lactococcus species or a Lactobacillus species. 5. The strain is Lactococcus latis.
ctis), The microorganism according to claim 4. 6. The trefoil peptide is TFF1.
The microorganism according to any one of 5 above. 7. A pharmaceutical composition comprising the microorganism according to any one of claims 1 to 6. 8. Including administration of the microorganism according to any one of claims 1 to 6,
A method of delivering a trefoil peptide to the digestive tract. 9. Use of a microorganism according to any one of claims 1 to 6 for the manufacture of a medicament for delivering a trefoil peptide to the digestive tract. 10. A method for treating gastric and / or intestinal diseases and / or disorders, which comprises administration of the microorganism according to any one of claims 1-6. 11. A method for treating a lesion caused by a gastric and / or intestinal disease and / or disorder, which comprises administration of the microorganism according to any one of claims 1 to 6. 12. Use of the microorganism according to any one of claims 1 to 6 for preparing a medicament for treating gastric and / or intestinal diseases and / or disorders. 13. A method according to any one of claims 1 to 6 for preparing a medicament for treating acute gastrointestinal tract diseases including acute colitis, acute recurrence of Crohn's disease and ulcerative colitis. Use of microorganisms. 14. A method for preparing a medicament for treating chronic and naturally recurring gastrointestinal tract diseases including Crohn's disease (enteritis regionalis) and ulitis colitis (colitis ulcerosa). Use of the microorganism according to any one of 1. 15. Use of the microorganism according to any one of claims 1 to 6 for preparing a medicament for inhibiting the formation of lesions due to gastric and / or intestinal diseases and disorders. 16. A method according to any one of claims 1 to 6, comprising transformation of the microorganism with a recombinant vector carrying a trefoil peptide coding sequence under the control of a suitable promoter and a suitable secretory signal sequence. Of producing the microorganism of. 17. A recombinant vector comprising a trefoil peptide coding sequence under the control of a suitable promoter sequence and a suitable secretory signal sequence. 18. The recombinant vector according to claim 17, having a nucleotide sequence represented by any of SEQ ID NOs: 1, 2, or 3.